Skip to main content

Full text of "MKULTRA DOC_0000173966"

See other formats

electric fish investigation 

Final Report 


8 March 1973 


























Mau timer cells 
Electro receptors 
Cranial nerves of Elect •ophorus 
Microelcctrode amplifier 

Microelcctrode amplifier: schematic diagram 
Loss in -dB with increasing frequency 
Loss in -dB with increasing repetition rate 
Device for sharpening metal electrodes 
Device for applying silver chloride 
Potentiometric device 

Device for measuring microelcctrode resistance 

Amplification factor, square wave 

Amplification factor, sinusoidal wave 

Recordings at different square wave 

Recordings at different square wave 

Adjustable Lucite Tray 

Instrumentation for Anaesthesia 

Solution 1:10000 in water on Stemarchus albifrons 

Sternarchus albifrons #7 normal electrical activity 

Stcrnarchus albifrons #7 in anesthetic tricaine methane 
sulfonate, 1 minute 

Sternarchus albifrons #7 in anesthetic tricaine methane 
sulfonate, 2 minutes 

Sternarchus albifrons H 7 in anesthetic tricaine methane 
sulfonate, 4 minutes 

Ste rnarc hus alb i frons #7 in anesthetic tricaine methane 
sulfonate, 5 minutes 

Sternar ch us alb ifrons, recuperating, 5 minutes 
































*1 M 

• *» 
*» O 

4 *> 



Stcrnarchus albifrons recuperating, 8 minutes 
Set-up for microelcctrode 

Sternarchus albifrons No. 1 just before anaesthesia 
Sternarchus albifrons No. 1 anaesthetized with thiopental sodium 
(2 min. ) 

Sternarchus albifrons No. 1 anaesthetized with thiopental sodium 
(13.5 min.) 

Sternarchus albifrons No. 1 anaesthetized with thiopental sodium 
(37. 5 min. ) 

Sternarchus albifrons No . 2 in aquarium water 
Sternarchus albifrons No. 2 in MS-222, 10 minutes 
Set-up for recording 

Close-up of the set-up for recording; 

Sternarchus albifrons after d-tubocurarine injection 
Ampullary, tonic electroreceptors 
Set-up for recording 

Anaesthetized, curarized Sternarchus albifrons 

Microelcctrode amplifier 


Synchronous tonic electroreceptor recording 

Nonsync hronous phasic electro receptor recoiding 

Gvmnarchus niloticus Cuv. #2 electric activity 

Gymnarrhus niloticus Cuv. #3 electric activity 

Gvmnarchus niloticus Cuv. #2 electric activity 

Gvmnarchus niloticus Cuv. #2 electric activity 

Gvmnarchus niloticus Cuv. #3 electric activity 

Gvmnarchus niloticus Cuv. #3 electric activity 
1 * ■ ■ ■■ — 1 ■ 1 — < 

Gvn narchus niloticus Cuv. #3 electric activity 7 
Gvmnarchus niloticus, Cuv. (baby) - transversal cut 


























Gvmnarchus nilotieus Cuv. - air bladder and spinal cord 


48 . 

Gvmnarchus nilotieus Cuv. - spinal cord 



Gvnmarchus nilotieus Cuv. - brain 



Delafield - Harris hematoxylin staining method 



Malapterurus electricus 



Malaoterus electricus on the scale 



Device for measuring the voltage 



nf tbR electric oraan of Malapterurus electricus 



Tonic, ampullary electro receptor 



Phasic, tuberous electroreceptor 



Tonic electroreceptor 



Phasic electroreceptor 



. Ampullary tonic electroreceptor 



Stimulus recording 



Tuberous phasic electroreceptor 




Stimulus recording 



tflcctrnrccentors of Gvmnarchus nilotieus 


64 . 

Underwater pattern recognition system 



Recording from an anaesthetized, curarized 
Stemarchus nlbifrons specimen 



Microelectrode recording 


' 67 

Gvmnarchus nilotieus - photo 



Gymnarchus nilotieus - placement of electric 
transmitting and receiving organs 



Fish Laboratory A . 



Fish Laboratory B 





Underwater pa Hern recognition system 




The electric organs of Stcmarchus albifrons , a South American fresh water weak 
electric fish, have been studied with emphasis on electroreceptors. The morpholog- 
ical and physiological characteristics of electroreccptors, ampullary and tuberous, 
a... e discussed. Special instrumentation required for the role of these electroreceptors 
in pattern recognition has been developed. 

We have recorded with microelectrodes the autonomous autorhythmic electrical activ- 
ity of the tonic asynchronous ampullary electroreccptors of the South American weak 
fresh water electric fish Sternarchus albifrons . We have also recorded the electrical 
activity from the phasic tuberous electroreceptors and of the synchronous ampullary 
electroreceptors of die same electric fish, Sternarchus albifrons . Preliminary meas- 
urements have been made . The electric discharge of Malapterurus electricus , an 
African fresh water strong electric fish, has been measured in and out of water. The 
autorhythmic activity of the ampullary electroreceptors lias been demonstrated. 

We obtained some specimens of the African weak fresh water electric fish Gymnarchus 
niloticus . They are supposed to be the most sensitive of all the weak electric fishes 
known. Together with two specimens about one foot long, we received a number of 
baby Gymnarchus niloticus about two inches long. The baby electric fish were in- 
fected with a Saprolegnia fungus and could not be saved, but we fixed a number of 
them in buffered formaldehyde and one of them has been cut and mounted in paraffin 
for histological studies of the electric organs. Preliminary measurements have 
been made on the communication capability of adult Gymnarchus niloticus . 

A study of the anesthetizing effect of tricaine-mcthancsulfonalc (MS222 = FINQUEL) 
on Sternarchus albifrons has been undertaken by plotting time for the anesthesia and 
recovery for different specimens. 


A study of an anaesthetic which docs not affect the electric fish’s electric organ 
pulse repetition rate is presented. 

Also, the effect of D-tubocurarinc and the counter -effect of neostigmine has been 
assessed for Sternarchus albifrons . Finally, some improvements in the micro - 
electrode recording instrumentation have been made. 

The electric organs of Sternarchus albifrons , a South American weak fresh water 
electric fish, have been studied with emphasis on electroreceptors. Recordings 
have been made from the asynchronous tonic as well as the synchronous tonic and 
the asynchronous phasic electro receptors. The electroreceptors are part of the 
complex lateralis line system of the electric fishes. 

The other lateralis line system sensory receptors, like mechanical receptors and 
displacement receptors, have been discussed as part of a general hybrid pattern 
recognition system of the fish. A passive hybrid underwater pattern recognition 
simulation system has been advanced. 

A simulating model concept could be established for underwater pattern recognition 
through electric sensory receivers and electric fields. More histological work is 
needed to establish the relationship between different electroreceptors and their 
innervation. This is also needed for a realistic simulation system of the underwater 
pattern recognition ability of the electric fishes . 


[n one of our previous reports, we described the morphology of the electric organ 
of Stcrnarchus albifrons , a weak fresh water electric fish from South America. 1 
We mentioned that the electric transmitting organ of Stcrnarchus is derived from 
nervous tissue and not from modified muscle tissue like the majority of other elec- 
tric fishes. This is malting Stcrnarchus different from other electric fishes: it 
has a very high signal rate and the signal is phase and amplitude modulated. 

The form, rate, and amplitude of the signals emitted by the electric fishes are as 

diverse as the forms and sizes of these fishes. Some of the weak signals are used 

to locate objects or animals in their environment, or for navigation, species recog- 


nition, and communication. The strong electric discharges serve for offense or 


defense. Watanabe and Take da investigated the effect of a-c current with a fre- 
quency close to the electrical signal emitted by Eigenmannia . When the applied 
pulses came within + 3 to 4 pps of the one emitted by Eigenmannia , the fish would 
change its rate by 4 to 5 pps in a direction which increased the pps separation. In- 
creasing the frequency of the applied a-c current in 1 pps increments caused the 
fish to shift its frequency correspondingly until it reached about 6 to 7 pps over its 
normal rate, when it would revert to its original rate. We obtained similar results 
in experiments with Stemarchus albifrons , but the applied a-c signal was within 0. 5 
cycle of the signal of the fish, demonstrating how specific the applied signal must 
be to elicit a change in the Stcrnarchus signal rale : +0.5 cycle is the range of se- 
lectivity of the fishes' electrical transmitting-rcceiving system. The fact that many 
fresh water and sea water electric fishes have never been studied may present some 
difficulties in obtaining special kinds of electric fishes, and their care may not be 
an easy task. There arc, however, enough species to enable many experiments. 


Very little is known about the electrical activity of marine electric fishes except 
Tor p C do and some rays. Narcine Barziliensisj s the only -known marine electric 
fish having two different electric organs . Bennett 4 studied the mode of operation of 
the electric organs of Ra ja eglantaria , a marine electric fish, and of the fresh water 
fishes lTypopomus and Stcrnopygus and compared them with Narcine, Mormyrus, 
Steatogenys , Gvmnorhamphychtis , Malapterurus , Gymnotus carapo , and Elec tr o - 
phorus electricus . 

The main objects of this study were the form, innervation, and physiology of the 
electroplates forming the electric transmitting organs. The electroplates of 
Hypopomus, Gymnotus carapo , Malapterurus , and some Mormyridae have the same 
surface area and produce spikes during discharge. The electroplates of Stcrnopygus 
and possibly Eigenmannia have two peculiar characteristics: there is a steady po- 
tential on which pulses are superimposed, and the resistance of the electroplates is 
similar at the peak or between the spikes . The electroplates are of the type with a 
slow depolarization. 

In Hypopomus , Malapterurus , and most of the Mormyridae , the innervation of the 
electric organs is through stalks. The stalks may serve to amplify depolarization 
until it would be able to invade the body of the electroplates. 

The discharge rate and duty cycle have been compared for electric fishes like 
Stcrnopygus (rate = 50/sec) and Eigenmannia (rate = 280'/ sec) with the Stemarchidag 
(max. 1500/sec). Compared with mammalian central nervous systems, peak fre- 
quencies of the electric organs are not greatly different. The Renshaw cell can 
discharge impulses at a rate of 1400/sec 5 , and neurons in the sensory path some- 
times produce bursts at a rale of about 1000/sec. 

hi an examination of discharge pattern and organ function studies, it was noted that 
Grundfcst^ defined two groups of electric fishes: those that emit signals at a constant 


rate, and those that emit pulses with a variable rate. For example, Gymnarchus , 
St crnopygus , and Eigcnmannia are in the constant rate group; Electrophorus , 
Gn athonemus petersii , Stcatogenys , and Hypopomus belong to the variable rate 
group. Bennett^ did not make any connection between the electric fishes’ electrical 

systems, their environment, and their behavior. No one investigated their evolu- 


tion, very little is known about their mating or birthplace, and no one has reported 
the breeding of electric fishes confined in water tanks. 

Sternarehus albifrons also has two kinds of electroreceptors: tonic and phasic, and 
they are autorhythmic. These electroreceptors are sensitive to movement and di- 
rection. The phasic electroreceptors seem to be related to informaticn regarding 
movement of objects near or around the fish. 

Accepting the principle of pacemaker activity in the brain, it seems that there are 
only a reduced number of command nuclei acting on the electric transmitting organ. 

It is also reasonable to assume that electrically mediated positive feedback must be 
present; chemically mediated transmission would be too slow for the repetition rate 
of transmission which can attain under certain circumstances over 1,300. 

Mauthner cells of lower vertebrates (Figure 1) can be considered single cells 

8 9 

command system for the, axial musculature on either side of the body ’ . In the 
hacketfish each Mauthner fiber activates the muscles depressings both pectoral fins 
and these cells thus constitute a bilateral command system for the depressor 
muscles^’ ^ For explaining the pacemakers action of the command nuclei in the brain 
of the mormyrid electric fishes a mutual excitation with positive feedback has been 
proposed. This theory would not work for the Mauthner cells. There is a crossed 
inhibition between the Mauthner cells in the brain of the goldfish and it could equally 
be effective in an electric organ system. There is a requirement of high speed 
''f transmission in synchronized systems like many of the electric transmitting 
organs. This has been useful in predicting sites where transmission has been 


cpti'c I'sbtfs 



1 o 0 « 


*4 On i 

^ I 

Figure 1. (a) Schematic drawings of Ijp location of the Mauthner cells (fi’om 
Furukawa and Furshpan ) 

(b) The principal circuitry involved in Mauthner x'cflex (only the Vlll-th 
nex-ve connections and collatcx-als deriving fx’om the excited Mauthner 
cell are shown). 



electrically mediated. Positive feedback may be a "sine qua non" requirement for 

the pacemaker nuclei of electric transmitting organs. There is proof of positive feed- 

12 13 

back in the mutual inhibition system of arthropod compound eyes * • 

Tin neural systems controlling electric organs have provided a large number of 
examples of electrically mediated transmission, which meets the functional require- 
ment for rapid communication between cells. This mode of transmission also proves 
to be able to mediate many functions often considered as restricted to chemically 
mediated transmission. The correlation between morphologically close apposition 
and electrotonic coupling was considerably strengthened by the work on electro- 
motor systems. This correlation helps to validate morphological identification of 
electrical transmission in other systems where electrophysiological analysis is 
not so simple. 

It is not known whether there is any relevance to higher systems of the organizational 
principles deduced from electric organ systems. The next level of analysis of the 
electric organ systems may be no easier than the study of less speciaEzed systems 
that are of more general interest. Some knowledge is being obtained of afferent 
pathways from electroreceptors in weakly electric fish winch have important inputs 
to the electric organ control system. Both operant and respondent conditioning of 
the control system can be obtained and conditioned response latency can be very 
short. It is not unreasonable that the complete neural pathway of the conditioned 
response could be obtained in these cases. The central connections are still 
minimally explored; one knows what goes in and one can go from the electric organ 
several synapses antidromically. The rewards for filling in the gap would be great, 
and prospects for at least some progress are bright. 

In Stcmarchus albifrons clectrorcccptors are distributed over the entire body. The 
l-iasic tuberous receptors are very numerous as compared with the tonic ampullary 
receptors (Figure 2). The density of receptors is greatest in the head region and 


falls gradually toward the posterior end. There are minor morphological subdivisions 

within the phasic and the tonic receptors, but no physiological correlations have been 

4 ,, . ,14,15,10 

yet obtained 

In Figure 2 the equivalent circuits of tonic, ampullary and phasic, tuberous organs 
is shown. Cross sections through the receptors are shown with the external medium 
to the top. The skin and walls of the receptor cavities are represented by lines, 
innervation of the receptor cells is indicated. 

The electroreceptors over the entire body are innervated by the anterior lateral line 
nerves, a large branch of which runs posteriorly to join the posterior lateral line 
nerve just behind the head 17 (Figure 3). The posterior lateral line contains only 
mechanoreceptive fibers which come from free neuromasts and canal organs^’ 

The receptor cells of tonic receptors appear to behave very nearly like linear 

elements; that is, their membranes have fixed internal potentials, resistances, and 

capacitances. They are, in a sense, electrically inexcitable, and they differ markedly 

in this respect from phasic receptor cells. There is evidence for chemically mediated 


transmissions at tonic receptors of gvmnotids . The morphological characteristics 

of the snyapse are those typical of chemically mediated transmission. A strong 

brief anodal stimulus produces an evoked response long outlasting the stimulus. Then 

there is a synaptic delay between the initiation of the impulse and the nerve impulse 

(between 0.5 and 1.5 msec). Mormvrid tonic receptors are similar to those of 

gymnotids and Gymnarchus niloticus tonic receptors are morphologically similar, 


but they were not physiologically studied 

The relationship between the different elcctroreceptors of Sternarchus albifrons in 
pattern recognition has not as yet been studied. The present study developed special 
instrumentation required for investigating the roles of these elcctroreceptors in 
pattern recognition and obtained preliminary measurements of electrical discharges 
^ rom Malantcrimis clcctricus in and out of water. 


Figure 3 

Horizontal projection of the cranial nerves of Elect rophorus . 
Note the small size of the brain. Some nerves are indicated by 
.the corresponding numbers. L. A. norvus lateralis anterior; 

. LP nervus latcral'is posterior; Oc-Sp, oeeipito-spinalis nerve. 













■ 's5 

Xn our experiments , the perception of objects by weak electric fishes by way of the 

discharge of their electric organs (transmitting and receiving) has been demonstrated. 

Other authors have shown that weak electric fishes* can be trained to distinguish 
between conducting and nonconducting objects placed in the water. 

For this land of reception, two possible modes of action at the level of specific re- 


ceptors could be proposed: the "pulse-frequency-modulation" and the ’’pulse- 
phase-modulation" . According to the first hypothesis, sensory information should 
be conveyed by the frequency of the sensory impulses dependent on the pulse of the 
electric organ discharge whereas, according to the second hypothesis, the time re- 
lation (the phase) between the electric discharge and the following sensory impulse 
would play an important role for the sensory coding. From our experiments with 
Stemarchus albifrons , a South American weak fresh water electric fish, we con- 
cluded that apparently neither of the above proposed mechanisms arc operating in 
this fish. The intensity of current flowing at the level of the receptor is coded by 
the number of impulses elicited by each electric organ discharge. Stemarchus 
albifrons can discharge at rates higher than 1000 per second and the sensory impulses 
of the receptors can follow' their discharge rate. 

In some mormyrids^ and gymnotids^, however, modulation of the relation electric - 
organ pulse -sensory impulse may be used for elcctrosensory coding. In mormyrids, 
also, the change in intensity of the electric field may be coded by changing the latency 
between the electric organ pulse and the sensory receptors’ impulse. In mormyrids, 
it may attain values up to 9 msec, whereas in gymnotids it has been found not to 
exceed 1-2 msec. 

It lias been previousty mentioned that in Stcrnarchus albifrons the cloo'.rore cep tors 
arc distributed over the entire body of the fish and that the ampullary ionic receptors 


are more numerous than the tuberous phasic receptors . We recorded -hen the auto- 
rhythmic electrical activity of the nonsynchronous tonic, ampullary o', uctrorcccptors 
°I Stemarchus albifrons. The impulses were irregular around a repo '--'.ion rate of 






























between 100 and 300, with an amplitude of around 2.5 mV. The impulse duration 
was around 200 microseconds. There are other types of tonic receptors which are 
synchronous . The phasic units are nonsynchronous. 

The nonsynchronous tonic receptors seem to react independently from the trans- 
mitting electric organ. They react to any objects brought near the fish at a certain 
range. The recording shown in our final report is made from such type of 

As previously mentioned, some fresh water wealc electric fishes have the ability of 
perceiving objects, their movement’ and direction, and also to determine some char- 
acteristics of these objects (such as conductivity). For this underwater pattern 
recognition, they utilize both their electric transmitting organs and their electro- 
receivers. Obviously some other sensory perception receivers of the lateralis line 
may be involved such as : free neuromasts and the cupula type of lateral line re- 
ceivers ; the auditive system may play a role also in this pattern recognition. Some 
of the electric fishes are blind or have vestigial eyes ; others have good vision and 
it is certainly used in the recognition process. 

It seems that a hybrid system made of a diversity of sensory receptors is used by 
the electric fishes to locate and identify objects and fishes of the same or different 
species and also prey or predators . They use little electrical energy for this and 
the distance involved is considerable — it may attain, under certain circumstances 
and for one specific sensitive species (Gymnarchidae) , over a mile. 

The strong electric fishes (Elcctrophorus, Malapterurus , Torpedo , and Astros copus) 
can incapacitate and Idll their prey with their discharge of the powerful electric 
organ used only for such purposes. If they have a detecting and locating system, 
they use a different low power electric transmitting than the main powerful electric 
organ (i.c. , Elcctrophorus) . The meaning of this is that there is no high power 
requirement for the pattern recognition system of electric fishes. The electric 


sensory receptors aro very sensitive, have a high discriminating capacity, and use 
an electronic processing system based on a multiple degree of freedom modulating 
and coding system; they are also jamming resistant. 

The importance of such an underwater pattern recognition system cannot be over- 
emphasized. The simulation and modeling of it can be achieved once the parameters 
of the different sensory receivers have been established, physical analogs derived, 
and models devised. 

By and large, the communication and coding system of electric fishes has been 


established and discussed . The antijamming capability of the system has been 


demonstrated through simulation . The electroreceptors of Sternarchus albifrons, 
a fresh water weak electric fish, have been studied, and for two different kinds , 
physical analogs have been proposed. 

Because of our previous findings that the anaesthetic "MS 222 (Fhiquel)" used by 
most researchers of electric fishes affects the frequency of the impulses emitted by 
the electric organs in a nonuniform way, we investigated a number of different 
anaesthetics and found one which does not affect the frequency of the impulses or 
their amplitude. 



The recordings of the autorhytmic electric signals from electroreceptors need 
special instrumentation. The signals are of low voltages: less than 1 mv, and they 
need a large bandwidth. The diameters of some of the electroreceptors are of the 
order of microns. There is the need for an insulated microelectrode with a tip of 
one micron, with a reasonable low resistance, possibly less than one megohm, 
responding from DC to several kHz with low distortion and enough rugged to with- 
stand some bumping by the fish. It has also to be of a nonpolarizable type. We 
developed a microelectrode with all these characteristics and it is described later 

in this chapter. 

In our proposal for the continuation of the investigation of electric fish we mentioned 
that we developed a sensitive low noise solid-state microelectrode d-c amplifier 
(Figures 4, 5). We modified them having now a Motorola MC 1531 input stage and 
a Motorola MC 1431 output stage. Graphs show the repetition rate versus amplifi- 
cation factor for square wave and frequency versus amplification factor for sinusoidal 
waveforms with a 100 kiloohms, 1 megohms, and 5 megohms input resistance 

(Figures 6,7). 

We give a description of the method used to produce a suitable microelectrode for 
recording electric signals from the electroreceptors of S temnr chus albifroii s^ 

llubc?5cscribed how to make coated tungsten microelcctrodes. Wohlbarsht, ct al, 

10 i 

described glass insulated platinum microclectrodes; Green and Grundfest, et al , 


used stainless steel electrodes. The steel and tungsten' electrodes have a fairly high 
resistance (20 to 100 megohms) and all, including the platinum electrode, are polarizable. 

Donaldson^ describes a multitude of microclectrodes such as silver-silver chloride, 
platinum-platinum chloride, and others. Silver-silver chlorides aic very convenient 


Figure 4. Microelectrode amplifier with one MC 1531 and one MC 1431 
operational amplifiers. 

Figure 5. Microelectrode amplifier with one MC 1531 and one MC 1431 

operational amplifiers: schematic diagram. 



electrodes but have a high resistance. In order to lower the resistance, it is 
advisable to cover the silver-silver chloride electrode after the electrode is 
insulated with insulex lacquer, with platinum black through an electrolytic process 
and after this to add a new silver-chloride coating. The tip of the electrode can be 
anywhere between 0.5 and 1 micron in diameter. 

Take a 50 ml beaker and fill it with cone. H Cl and cover it with Xylene (about 
1/2"). Use a carbon rod for one electrode (spectroscopy carbon rods are suitable) 
and connect it to a variable source of A.C. current from a variac and a bell-type 
transformer (app. 7 to 12 v). Connect the low voltage leads to an A.C. voltmeter. 

Use about 5 to 6 volts to sharpen the silver wire #36 gauge (any necessary length) by 
moving it up and down for about one minute (Figure 8). Decrease the voltage to 
between 2-3 volts and move the electrode rapidly for 30 seconds up and down. Have 
50 ml beakers filled with: (1) saturated sodium carbonate solution (NUgCO ), 

(2) acetic acid 1% in H O (CH CO) - O, (3) Ethyl-alcohol 200 proof, (4) Xylene. 

Move electrode after sharpening process from No. 1 through No. 2, 3, and 4, agitating 
a few times the electrode in the liquid. Check under microscope with a microfilar for 
sharpness; if not sharp enough, operation II (2-3 volts) and. the cleaning from 1 to 4 
should be repeated. 

If sharp enough, take a 50 ml beaker and fill it with Na Cl 1% solution in distilled 
H^O. Use a silver wire (No. 18 to 22 gauge) as an electrode (cathode) and connect it 
with a D.C. source (power supply) of between 1 to 2 volts. The positive end should 
he connected to the microelectrode. Hold it for 30 seconds in the H Cl solution. 
Heverse twice the polarity for the same amount of time (Figure 9). Wash the 
electrode in distilled water for two minutes. Insulate the electrode with insulex 
lacquer. For drying, set the microelcctrodcs with the tip up. Dry for 24 hours. 

The lip should be clean of lacquer for 10 to 30 microns. Take a 50 ml beaker and 
kll it with a 1% chloroplalinic acid. Use a No. 18 or 22 gauge wire as a cathode 


Figure 8. Device for sharpening metal electrodes. 

Figure 9. Device for applying silver chloride on the lip and surface 
of silver or silver chloride platinized electrodes. 


•onnected (to the negative) to a D.C. source of 15 volts. The microelectrode should 
£ connected to an anode (positive) of 15 volts, in series with a 1 Megohm (1 W) 

•csistor. Pass current through the electrodes for 15 to 30 seconds. Wash the 
•lectrode in distilled water for a few minutes. Use the 1% Na Cl solution with a 
.jlvcr wire, gauge No. 18 to 22, as cathode, and the 15 volts D.C. source in 
.-cries with the 1 Megohm resistor for depositing a silver-chloride coating on the 
•ip of the microelectrode. Twenty to 30 seconds will be sufficient. If bubbles come 
off from any other part than the tip, it means the insulation is not good and should be 
redone. Wash the electrode in distilled water for 10 to 15 minutes. Store it in dark 
rontainer filled with Ringer solution. 

The electrode has low resistance (from 100k to 800k depending on the tip), is non- 
•jolarizable and produces very little distortion from D.C. to fairly high frequency 
(over 1000 Hz) (Figures 10,11). 

In Figures 11A, 11B, 11C, and 11D, the performance of the microelectrode amplifiers 
is shown. 



Figui'clO. Polcntiometric device and 
resistance substitution and series 
resistance box for measuring micro- 
electrode resistance. 

Figure 11. Device for measuring mici'o- 
elcctrode resistance with the aid of the 
polentiomctric device. 






Fitrurc 11A. Amplification factor versu: 


V «gr? 

kii£* 'iifiLSf’ 

*J -.H'^rl^? 7 ?/rF> • ' > e r***?#* 

■'.•'.:V‘i-"'»lL-''.i -.ii.-*-- - : >*<' •. .•«•■'•• ■■•■.'*'.■ 

• ;: -^v 

!£• *’ ■'..•v’riy' ''•«u*'' i *jfe? : W V*. /V */> _ 7 

; J* ,' ^n' •'* / 1"‘*V^- "\ ••' * ■- . * * «* /> 7 * 

; • V -TV. *• V*.*- >• , •’* . • -'* / ', V - 7 * . 

*. y*.;u. •••:»*• v'.-v 7 '*'*'*- %••.:’ V.;^.v :»*«.'.• >:/*V 

■ \ • - ■ •-* - *i 

*•• .V’. ’-j7 -*** ';■? vl'-d'*"- ■’.*r'77 /•- . . V . 

— ‘ *! r ‘' * * ' -■* . .1 '• ~ : ' /’ .' 

v.«- ; ♦ . ' • 

:*■: :'■ j F- ‘r- 

■'' ,Lw '- " 

■ - -:-■!-..■>»< >->** *»'. - •' '•' 

••• -' •.•v..-'-:'-.vr : : i r2 r >™.-..“!.. : r > :<;'.* • •.■•/ ■ 

7V\3 v T: :: >;;^v!^7^si •' -,7 

■■ ''. ► -’V '- ,v-‘ ' ‘••Tiy.- ii /«*‘ '/' 

■'• f. * V. *' ■ . 4><‘5> V 7‘ * *• • 

. .• V*/»V*. k - V* /‘■V 

1 megohm. 


Flg.U-D according at dMcre* 

frequencies with Ihc double stage MC 1531 microciecnoa i 
resistance 5 megohms. 





j A C ommonly used anaesthetic for fishes and cold-blooded animals, urethan (ethyl 
* arbamate = C H NO ) has been found to have carcinogenic properties by Wood 
j ^ gan ^ Cowen 3 ^. An editorial comment accompanying Wood’s report indicated 
1 that the substance named MS-222 (today called Finquel) might be a suitable substitute 
| for urethan. MS-222 (tricaine methanesulfonate = c 10 H 15 NO 5 S) was discovered by 
i M. Sandoz during Ms search for a reliable synthetic substitute for cocaine (benzoil- 
j methylecgonine = C u H 21 N0 4 ) for a local anaesthetic. 

j since Wood’s and BaH and Cowen’s reports, MS-222 has become routine for anaesthe- 
j tization of fishes to facilitate the handling of both marine and fresh water species. 

1 The toxicity of the drug to some species of fish was determined by Marking . Walker 

| and Schoettger 37 measured its residues in various tissues of salmonides and its 

1 cfficacity has been investigated by Schoettger and Julin 38 . The chemical and anaes- 
| thetie quaUties of MS-222 are mentioned by Klontz 39 , and details about the chemistry 

I 40 

I can be found in the Merck Index . 

| In the last years (1970-71), an agreement lias been made between the Sandoz Pharma- 
j ceuticals , Division of Sandoz, Inc., Basel, Switzerland, and Hanover, N.J., USA, 

1 and the Ayerst Laboratories, New York, N. Y. , that the latter should produce and sell 
j the MS-222 (tricaine methanesulfonate) in the United States under their own brand 

| name of "FINQUEL” . 

\ In most of the studies on electric fishes, MS-222 has been used as a general anaes- 
4 • aa 42 « <• 

§ thotic by the investigators. Szabo , Hagiwara, Szabo and Enger , Enger and 

1 Szabo 43 , Nobuo Suga 44 , and many others used MS-222 to anaesthetize electric fishes 

| during surgical procedures and subsequently to record the electrical activity cither 

| from' the electroreccptors or from the nerves connecting them with the central 

■4 " 




vus system. No mention has been made of the effects Of the MS-222 on the fre- 
quency and amplitude of the electrical signals as related to concentration and/or time. 
gullock 45 mentioned that Hvnonomus occidentals , a South American fresh water weak 
i c ] C ctric fish, with a normal repetition rate range of 25 to 90 per second, would lower 

j its frequency to below 16 per second only under anaesthesia. Under deep anaesthesia, 

! the fish may stop abruptly its electrical activity. By reducing the level of anaesthesia 


or by stopping it to let the fish recover, it will also abruptly resume the normal 
; c |ectric activity. In our experiments with Sternarchus albifrons , a South American 
j fresh water weak electric fish, we found a gradual tapering of the repetition rate of 

I t jj e electric signal with the deepness of the anaesthesia and a gradual increase of the 

repetition rate with the recovery. By monitoring the effect of Finquel (MS-222) 

I anaesthetic on the electrical activity of Sternarchus albifrons , it has been observed 

j a fast and significant change in the repetition rate of the electric signals. A decision 


t was made to study the effect of different anaesthetics on the electrical activity of 

> different species of electric fishes. 




1 * T . 

I The anaesthetic agents of choice were: 









A specially built 00 cm tray made of lucitc which could be adjusted to the size of the 
fish lias been used to check the effects of anaesthetics on the electric oigans of elec 
trie fish (Figure 12). The tray is provided with fittings for the rapid discharge of 



^aesthetics or water and a constant aeration of the solution is possible if necessary. 

It also has at every centimeter distance embedded stainless steel electrodes con- 
noted on both sides of the tray with the exterior and to contacts . The electrodes 
corresponding to the position of the head and tail of the fish were connected through 
a high-gain, low noise amplifier to too oscilloscopes Tektronix (one for photo-taldng) , 
a Junter and an FM tape recorder (Figure 13). The tray with the fish was located in 
a floating screen room and all the instrumentation grounded to the screen room was 
located in the laboratory outside the screened room. A Incite cover on the tray pre- 
vented the fish from jumping out, different holes served to pour in water or anaes- 
thetics^ put in tliermometers / and to let excess air out. A d-c (battery) operated high 
intensity lamp was used to illuminate from the top the fish and to furnish enough heat 
to hold the temperature constant during experiments. For the experiments, the 
fishes- own aquarium-water was used at the beginning of the experiment and the 
same water was used for mixing in the anaesthetics. In this way, the temperature 
of the water and anaesthetic solution was easy to keep to the same level as the normal 
temperature of the water in the aquarium. The pH of the aquarium water at the be- 
ginning of the experiments and also the pH of the anaesthetic solution were measured 
with an expanded precision type of pH meter. The pH meter has been calibrated 

before each experiment. 

Tentative measurements and observation of the action of the anaesthetic have been 
made first on goldfish and then on one of the electric fishes before another specimen 
has been selected for the experiment. For each experiment with one and the same 
anaesthetic, five specimens of sternnrehus albifrons have been used. The specimens 
of swnnrnhns albifrons , a fresh water South American stemarchid weak electric 
fish, have been in our laboratory for over one year and they were all healthy and 
varied in weight from 14 to 30 grams. Gvmnarchus nlloticus, a fresh water African 
gymnarchid weak electric fish/ has boon kept for over six months in our laboratory. 
There is another specimen just received. There arc also two Onalhonomus poters ii, 
fresh water African mormyrid electric fish, being in the laboratoiy for over too 

Figure 12. Adjustable Lucite Tray for Anaesthesia Experiments. 

| Figure 13. Instrumentation Used for Anaesthesia Experiments. 

! i 




i The fishes have been weighed before each experiment and put in the experimental 

| tray in their own aquarium water. The electrical activity of the electric organ has 

) been monitored, recorded with the FM magnetic tape recorder, its amplitude meas- 

j u red bn the calibrated oscilloscope, and a photo taken. After a few minutes, the 

j wate r was discarded from the experimental tray and the anaesthetic was introduced 
\ with a funnel through one of the holes in the tray cover. The time of introducing the 

I anaesthetic has been marked with the aid of a timer and also recorded on the magnetic 

I tape and in our records. The effect of the anaesthetic on the fish, its behavior and 

§ re spiration were constantly observed. The electric activity has been monitored and 

l from time to time a photo has been taken from the oscilloscope. The moment in 

I which the fish was anaesthetized completely has been recorded. The fish respira- 

tion and electric activity (amplitude, wave form and repetition rate) were constantly 
observed. If necessary, the anaesthetic has been immediately discarded and fresh 
aquarium water has been introduced in the experimental tray with adequate aeration 
for the fish. This moment has been recorded and the recovery time of the fish has 
been marked. The electric activity also lias been monitored. When the fish was 
considered completely recovered, it was returned to its own aquarium. Sometimes 
because of the long time a fish was anaesthetized, it has been returned from the ex- 
perimental tray in a net floating in its own aquarium with adequate aeration with 

bubble stones under the net. 

Experiments were performed on five specimens of Sternarchus albifrons (No. 2, 3,4, 
6 and 7). In order to assess the effectiveness and dosage of the "MS 222 Finquel” 
tricaine methnnesulfonate, we recorded the electrical activity before, during, and 
after anaesthesia. The frequency of the electric organ will drop immediately after 
adding the tricaine mothanesulfonate to the water in a special tray, provided with 
stainless steel electrodes set at a distance of one cm from one another over the 
length of the lucite tray. The fish were restricted by a partition and a "U" shaped 
lucite device placed on top of the fish. Figure 14 shows the decrease in frequency 






£ © 
t £ 

» i? 

© >5 
to jj 

'S 1 

O fi 

© a 
■h ° 

{S !h 
O O 

a s 

■C © 

© O 
P. 1* 


fc *3 

© § 


53 ej 

§ g 

fa £ 

« w 

ts 3 ■ 

I § 

! 5 2 

; © © 

J O p. 

1 g g 
: * 8 

a « 

*3 to 
W g 


^ ~>tai & 1 St lli 1 i. fe H£M aik-i jjfp 

U2 o 

easindiui ITOixpoH J° OTOI dotmodaa 

wifll time when the fish has been kept in a 1:10000 MS222 anaesthesia solution in 
wter and the return of the impulse rate after the solution has been exchanged with 
fresh water . The electrical activity of the electric transmitting organ was recorded 
on a Hewlett-Packard four-channel PM magnetic tape recorder to be played back and 
emdyzed later. Figure 15 shows the electric activity before anaesthesia; Figures 16, 

17 , 18 and 19 show the electric activity during anesthesia; and Figures 20 and 21 
Show activity during the recuperating process. 


From seven anaesthetic agents we tried, six were inadequate because the repetition 
rate of the electric signal ms affected. Only the thiopental sodium Abbot (pentothal 
sodium) does not influence the electric activity of Stemarchus albifro ns- 

The thiopental sodium has been cheeked on five different fishes (same species). It 
has a very fast effect in a dosage of 1:10000 in water. The fish, after it has been 
anaesthetised, would remain as such for 2 to 4 hours lying very quietly. It recovers 
completely and the anaesthetic has no ill effect on the fish. We anaesthetised them 
repeatedly and after six months' time they are doing well. After the fish has been 
anaesthetised, it has to be put in aerated fresh water where it can remain for hours. 
Figures 23 , 24 , 25 and ?6 show that the repetition rate of the electric signal has 
not been affected in over 40 minutes. The only change was produced by a slight de- 
crease in water temperature (from 720 pulses to 700 pulses) of about 0.4 C. 

Stornarehus albifron s is very sensitive to changes in water temperature. It tvrll 
increase the repetition rate of the electric signal for an increase in water temperature 
md it will decrease the repetition rate for a decrease in water temperature. For 
every degree Centigrade, it may change the repetition rate by about 50 to 80, de- 
pending on the particular fish. 

Tluopontli sodi-m or Sodium 5-eth y l-5-a-methyltatyl)-2-Uuobarbiturate has the 

chemical formula c u H 17 NaN 2 0 2 S ' a molooular wcisht ° £ 2M ‘ 33 an<i U 1 ycUo "' 1Sh ‘ 

.. cfo vnnvchus albifrons #7 normal elec 
^thnty. Water temp7 24.2°C, time: 

ri cc/cm, ? niV/cm. 

Fig. 16. Sternarchus albifrons #7 in anes- 
thetic tricaine methane sulfonate 1:10000. 
Water temp. 24.2°C, time: 1 msec/cm, 

5 mV/cm, 1 minute. 

| i Srerrnrchus nlbi frons "7 in ancs- 
l trier.:-.'.- methane sulfonate 1:10000. 

| *• rtsir.r. time: 1 msec/cm, 

} "'•'/era, 2 minutes. < 


Fig. 18. Sternarchus albifrons "7 in ancs- 
thetic tricaine methane sulfonate 1:10000. 
Water temp. 21.2°C, time: 1 msec/cm, 

5 mV/cm, 4 minutes. 

A ^ 

3 sternarchus albi Irons #7 in anes- 
•i'c tricaine methane sulfonate 1 : 10000 . 
ter temp. 24.2 C ‘C, time: 1 msec/cm, 
-V/cm, 5 minutes. 

Fig. 20. Sternarchus albifrons recuperating 
in fresh water. Temp. 24.2°C, 5 minutes 

• * /. W - 

’•• Sternarchus albifrons rccupei*nting in 
water. Temp. 24.2°C, 8 minutes. 

Fig. 22.. New set-up for microclectrodo 

rs r> 

- Figure 23. Stemarchus albifrons No. 1 just before anaesthesia with 
thiopental sodium. Electric signal rate: «710. 

Sweep 1 ms/cm. Gain 10 mv/cm; Water Temp. 22.4°C. 

: ; rmw mimi 

\; : -*---.i d/.. -M-.V/.-a W. -rA.r. 

"••i > ;; : : 11 <i/ 

i - i. . . j t . •. * k • . ■ "T? - '*-*«• . ? r ■**> * 

' • - S’- . .51;. .--iff ■ > r ' ■■.•’. -ri '. ; '3«. '■ >' . 

Figure 25. Stcraarchus albifro'ns No. 1 anaesthetized with thiopental 
° sodium 1:10000 (13.5 min. ). Electric signal rate: « 700. 
Sweep 1 ms/cm. Gain 10 mv/cm, Water Temp. 22.3°C. 

* _- I • . » • v .■« *.* >- r * - =***.. 

* N : • 
-.v ; ‘ 

Figure 26. Stcraarchus albifrons No. 1 anaesthetized with thiopental 
sodium 1:10000. 37 . 5 min. in fresh water. Electric signal 
rate: « 700. Sweep 1 ms/cm, Gain 10 mv/cm, Water Temp: 22.3°C. 

white , hygroscopic power, soluble in water ahd alcohol and is a strongly alkaline 

, . 46 

solution. Tlie Abbot preparation is of nonhygroscopic crystals . 

Tubocurarinc chloride can be administered by intra -abdominal injection. There is 
no interference between tubocurarine and thiopental sodium. 

The stability of thiopental solutions depends upon several factors, including the 
diluent and conditions of storage. It is recommended to keep them under refrigera- 
tion and tightly stoppdred. 

"Finquel" or "MS222" is a meta -amino-benzoic-acid-ethyl-ester in the form of tri- 
caine methane-sulfonate and has the chemical formula C 10 H 15 N °5 S with a molecular 
weight of 261. 31 and is produced as fine needles, soluble in water. It is slightly 

acid and is stable to boiling. 

Finquel 1:10000 in aquarium water would affect the repetition rate of Sternarchus 
albifrons and make it decrease in 10 minutes from 780 to 440 (Figures 27 and 28). 



FWr e * T. No . 2 in Acpanum w-~ • 

Electric Signal Repetition Rate: « 780. g 

Amplitude 25 mv, sweep 1 ms/cm, water mp. • 

„„„ *. 

Solution 1:10000 m Water. Electric -i D r 

After 10 Minutes in ^ temp. 22.4-C.-8/31/72 

Amplitude 38 mv, sweep ficq. , 

, xvtm MK,i A , a-j.woT5«af W* ■«■>*" 


four specimens of Stcraarchus albifrons have been used for establishing methods to 

record the autorhythmic activity of their electrorcceptors (Figures 29, 30). The 

fishes have to be anaesthetized and curarized in order to avoid twitching of the muscles 

during microelectrode recordings. We tried the effects of d-tubocurarine on 


s tornarchus albifrons. . Hagiwara. et al , recommended 0.05 to 0.1 mg curare/fish 

and Enger and Szabo recommended 0. 03 mg d-tubocurarine/g fresh fish weight. 

? Both used (MS 222) tri caine methancsulfonate for anaesthesia (1:150,000). We found 
I that the quantities given did not correspond in our case. 


l The weight of the Stcmarchus specimens varied between 16 g and 19 g. One specimen 
(16 g) received 0.5 mg d-tubocurarine intra-abdominally. The fish was paralyzed in 
one minute. Electrical activity of the main electric organ subsided after 20 minutes 
and the fish was dead after another ten minutes. All the time the fish was kept in a 
4000 ml beaker with medicated aquarium water at a pH of 7. 0 and a temperature of 
23.0°C. An aerator stone provided the necessary air. 

A second specimen of Sterna rchus (also 16 g) has been anaesthetized with MS 222, 
1:25,000. One gram of MS 222 has been dissolved in 1000 ml distilled H O as 
stock solution. Then this was diluated to the proper amount in a 4000 ml beaker by 
adding aquai'ium water with merbromine and acriflavine added as disinfectants. 

The MS 222 dilution of 1:150, 000 would not affect the fish in over one hour. The 
dilution of the MS 222 was reduced to 1:25,000. After 20 minutes in the MS 222 
solution the fish was injected intra-abdominally with 0.05 mg of d-tubocurarine 
l mil solution. The fish was paralyzed in three minutes. For one hour it gave a good 
strong electric signal of the maip electric organ, after, this gradually it diminished 
in strength and in another hour the fish was dead. 

,WS - sti rst anaesthetised in 

. ott „[ Stcrnarchus (18 S> «» Momto all 5 "« h # * 1 “ 

-* r;;:^ -« «. * - «•£ £lsh ^ «. *- - * : 

500 £0 " (Figure 31)* Tnrrbroiuiue 1 acr 

05 **" Mrated riUceP-s has 

;ial recording ^cording bo» ^^uver cWo^e 

dne «* «* 2M ' ' i ec trode * a. *« Wter “ tor figure *«• ®* 

, fter 30 minutes and treatment as the 

,« tank alter » ^ sa me tied. 

. (19 s) receU t-rav water » a 

■ „„ o£ SternarslBS- (1 8 , . lsc trode in tte tr J 

M0tte r fourth spec«n o£ using the neutra ^ t0Jlic , 

third specimen* *to *. diameter P tri0 organ signal 

silver-silver -**- ^ ,,, *, time the ^^tor. 

Hilary elOCtr0re ° o e ; aings J the authorhythmie aettm almoS t instantly, 

did not mash the reco Mrne d to Us tank an since then (US' 110 

Ub> the previous P 

, . . shielded room ti 

-~~:zzz~‘*"r~jzz- - ~ - 

waters were trim mic roseopo support, 

Other equipment, such ns 

o3 i 3 Sss s^S^ w ^ naaat 

s " 103 

trurc 32. 

. of g»^y »nrc*hus_ 

Ampullae l»"' c ^’‘^'''"“irTrc ^niiiilhuy receptor 
The dark point:, ' UL 


din «- autorhythmic activity oi ^* lver - 


iplifiers, etc. , was connected to one point ground and to. the Tektronix T22 modified 
tferential amplifier ground. The microelectrode amplifier and support was changed 
om the integrated circuit model used before to an electrometer tube type and a three 
•ansistor amplifier which is less sensitive to the change in impedance produced by 
light movements of the fish than the previous one. The microelectrode, a silver- 
ier -chloride, platinum, silver-chloride electrode of 0.5 micron tip diameter, was 
positioned on the electric receptor inside the grounding loop. Figure 22 shows the 
recording setup and Figure 35 the schematic of the microelectrode amplifier. 

At the same time, the electric activity of the receptors was displayed on one of the 
traces of a dual scope. The activity of the electric transmitting organ was recorded 
with two carbon electrodes placed at the end of the tray and connected to another 
Tektronix T22 amplifier and displayed as a second trace on the scope. A Hewlett- 
Packard four channel FM tape recorder was used to record the electrical activity 
of the electric receptors on a magnetic tape. It can be played back and analyzed at a 
later time. Photos were made during the recording. Figure 37 shows the activity of 
a synchronous tonic electroreceptor (in a previous report we mentioned that we re- . 
corded from a nonsynchronous tonic electroreceptor), and Figure 38 shows the elec- 
tric activity of a nonsynchronous phasic electroreceptor. After finishing the experi- 


ment, the fish was injected with neostigmine methylsulfate 1 :10 to counteract the 
effect of the D-tubocurarine. The fishes recuperated in a few minutes and are doing 

Another experiment was performed with two Gymnarchus niloticus, Cuv. Each of 
these fishes was placed in 25 gallon water tanks with lucite trays raised to approxi- 
mately 4 inches from the top. Gymnarchus niloticus is an air breather and if left in 
a deep tank must expend excessive energy to swim to the surface for breathing. The 
tank is kept clean by lucite plates provided with holes to let dirt fall to the bottom 
where it can be vacuum-cleaned very easily through special holes. The tanks of 

. • {V 


„, 0 no-*^ rMro9U,i ' r ' 


ffigli impe^ ce ® 

A ti"- '*• . 1 r - • * *• ■ -iJi k~— J LS-~rr*+ r T&-?~~ ‘..; j 

-. - .,« pi,:.:?. Ki'i '. -P * / 

-. v r. iv.^. ^j-J,-; .• £a* 

Flg . 37. SterrgESS^gg,^' MiWO-^ 

chronous tonic x msec/cm, 100 tcl 


,, lifro nS Ko " 51 ' n ' 

!». SSHSSaffl^^*,,. »llcro- 

•m>® P"“ ic ° ° 2 n ,scc/cm. 1W ""/ cl ' 

•i Tn rtfi rccorain 0 , 



nnarchus niloticus are approximately three feet apart. . The covers and outside 
.magnetic stainless steel frames were all grounded and so was the tank through 
•bon electrodes placed in the filters. 

to carbon electrodes in lucite tubes (with holes) were placed at a distance of 15 cm 
jm each other alongside the fishes. A third electrode was placed either midway 
: tween the recording electrodes or on one of the ends of these electrodes . The first 
;o electrodes for each fish were connected with the T22 Tektronix amplifiers and 
xmected to two different Tektronix scopes. The third electrodes were connected 
) a DPDT switch which could actuate also a battery to raise the lower trace of the 
cope when the two electrodes in the two tanks were connected and hence a communica- 
ion link was established between the two Gvmnarchus to study the effect of social 
nteraction and communication between them. 

n Figure 39, the normal electrical activity of the electric organs of Gymnarchus 
[o. 2 and in Figure 40 the electrical activity of Gymnarchus No. 3 can be seen. 

'igures 41 and 42 show that Gymnarchus No. 2 almost stopped the electrical activity 
jr ten to twenty seconds . Figures 43 and 44 show the modified activity of die im- 
ilses from Gymnarchus No. 3, and Figure 45 shows die modified activity of Gymnarchus 
o. 2. When a metal rod was introduced in the tank of Gymnarchus No. 3, Gvmnarchus 
j, 2 would nervously move back and forth and eventually attack the electrode connected 
th the other fish. This is only die beginning of studying die communication between 
o Gymnarchus niloticus . 

e Gymnarchus niloticus baby was sectioned and fixed in buffered formaldehyde (10%) 

• about one month. Then it was decalcified with Kristensen’s decalcifying solutions 
24 hours. After this, it was dehydrated using ethyl alcohol, toluene, toluene with 
affin, and finally embedded in degassed paraffin with 5G°C melting point. 




2 msec/cm, i ™ 


>** *%*?. vlIT- \" i s* x^AO £h&WI 



tiVily ° ( ?o°ctel“S oo, mooted through 
^ teen clcctucai > switch with 

.* electrodes and a ‘ w scc/cnv, 

,*„£ another CvnumrolnS. 


Fig. «»• ... 

Electric nctmty c ‘ conn cdcd throng, 

the to* has been elect • s%vi tch "f' 

carbon electrodes end n "' m scc/cm: 

Z tb* mother 
1 mV/ cm. 

« r* 


- 44. Gvmnarchus niloticus Cuv. #3. 

Icctric activity of the electric organ when 
ic tank has been electrically connected 
rough carbon electrodes and a ware and 
-dtch with the tank of another Gvmnarchus . 
msec/cm; 1 mV/cra. 

Fig. 45. Gvmnarchus niloticus Cuv. #3. 
Electric activity of the electric organ when 
the tank has been electrically connected 
through carbon electrodes and a wire and 
switch with the tank of another Gvmnarchus . 
2 msec/cm; 1 mV/cm. After one minute, 
the fish switched to its normal activity but 
with a smaller amplitude. 

An A. O. Spencer microtome lias been used to cut 10 micron slices. They were 
stained w’ith Hematoxylin-Eosin and mounted with Permount on microslides . There 
are about 27 microslides with four to five serially cut transversal slices numbered 
from the head in the direction of the tail: 1-1, 1-2, 2-1, 2-2, . . . , 7-3, 7-4. A few 
photomicrographs are shown as Figures 46 , 47, 48, and 49 . The interpretation of 
the histological preparation will be made at a later date. (Figure 50) 

Malaptcrurus elcctricus , the electric catfish (Figure 51) is different from the other 
dectric fishes because it docs not obey Pacini’s law. All the other electric fishes 
bey Pacini’s law, according to which the innervated faces of the electroplates be- 
ome negative during the discharge, whatever the anatomical orientation of the organ, 
his fact is due to the unique anatomy of Malaptcrurus . The electric organ of 

•alaptcrurus forms a sort of loose jacket around the fish, instead of being embedded 

♦ J 

v( l>>vv» • 

/■.l *7 l.f'^-‘ .<• ■.:- 1 ' • • *?>: .:. j«.'i 

*'5V * ' _ " >, -•»• w'; ^ , k/ ( ; 

*'2, t ’ — . r* * / V - * -, ' " *' ' 

u;v~ v/ v -; V '•,**'><> ,V 
V 1 V „S ; 

, \ - • •.•.■* .ti : ■ :•■•• . - 

=:. -V, ■. • t-> v '♦. » ■ ... •/■,;•> 

. •. 1 ■• ■•* f 

■~\- ■ iryr\ un. 

Vi • ‘ 

• L\- * k '>. *..■*. .z \.;v 
. ' ' 

. I & 



> Vv^-'n V - ^’v- v 

V' & V ' w- • 

'V'. ■ -V • 



j, U 

4 ,t» r» 


v w* , • 

V^A -i 


a-.*/; ,1 

' fe . 


‘■tc. a 

' ^ ' . 
!/; ! 


i*1 , 

IP tp 

t «• . * % - »• 

Figure 46. Gvmnnrchus niloticus, Cuy. (baby) 
Transversal cut through the last 1/4 in the direction of the tail 

. i fl r i,..rr. 

Piva H /■**•* 

i i » 

vmnrchus m'loticus Cuv._, baby 
s). Air bladder and spinal cord. 

, graph Nikon Phase -Interference 
°x 80. Fixed: Formaldehyde, 
c ifying^Hem atoxylin-Eo sin .sm > » • 

Fii r. 48. fivmnarchus mloticus Cm\ , baby 
(L 2 months). Spinal cord. Photomicro- 
graph Nikon Phase-Interference kbcroscope 

x 200. Fixed: Formaldehyde, 10*.. Dc “ 

calcifjang^JHcmatoxyli^-Eosin^m . 

nn s niloticus Cm^ , baljy 
Brain. Photomicrograph 
-Interference Microscope x 
Formaldehyde, 10?o. 

Ilematoxlin-Eosin J V-n . 


thin the body muscle as in all the other electric fishes . The electric organ, depend - 
on the fish's size, forms a sheath from 2 to 10 mm or even thicker, in which the 
;ctroplates are stacked in the longitudinal direction arranged in a somehow non- 
dcrly fashion, contrary to the other electric fishes. The electroplates arc disks 
about 1 mm diameter and 20 to 40 microns thickness and derived from myeloblasts, 
the center of the caudal face of each electroplate, a thin stalk arises from a complex 
agination. The end of this stalk makes contact with the branches of a single myelinated 
•ve fiber arising from a large nerve cell situated in the gray matter of the spinal cord 
ween the levels of the first and second spinal nerve roots. As it can be seen on 
ure 51, the nerve trunk which is heavily myelinated is easily identified. The way 
vhich the electric organ is innervated plays an important role in the synchronization 
Hianism of the discharge. 

Malapteruri dae discharge consists of a long train of 15 to 30 monophasic pulses 
to 2 msec duration in a total time-interval of approximately 100 milliseconds. 

ix to seven inch electric catfish could discharge impulses of approximately 200 V. 
ny surprise, Dr. Earl Harold from the California Academy of Sciences and Stein- 
It Aquarium in San Francisco, told me that the two very large (approximately 2 to 
)ot) electric catfishes which the aquarium had did not discharge more than 100 volt 

surprising that the Malnpteruridao we checked could put about 0. 5 w/hr of energy 
;ach gram of electric tissue of the electric organ. It is our intention to investigate 
fact more closely. The electric discharge of three strong fresh water electric 
s Malaptcrurus olcctricus weighing between 55-65 g was measured when in the 
r and out of water in the special tray with contacts. They discharged between 
30 volts in water and between 150-190 volts out of water. The discharge consists 
rsts of 5 to 7 impulses of II 5 to 2 msec duration (Figures 52, 53, 54). 


t emrus , «lectrlcus ^ The Nile Electric Catfish. 
(Shown at 1.5 Scale) 

Figure 52, 

1ft In pterurus clcctricus . African strong fresh water 
fish on the scale: weight 61.5 g. 

Figure 53. Device for.mcasuring the voltage of the discharges of 
V Malaptcnmis cloctrirns. 



Figure 54. Discharges of the electric organ of Malaptcrurus electricus 

we have now in the laboratory. Vertical: 1 graduation = 50 V. 
Horizontal: 1 graduation = 5 msec. 

In our synoptic Table No. 1, the past achievements and results of the study are men- 
tioned, the proposed continuation work is shown, and the future objectives are delineated. 

The multinrodulation-multicocling communication system of electric fishes served as a 
nodel in the development of a communication technique to resist jamming. 

lie physical analogs of a phasic and a tonic electrorcceptor have been established for 
re Stemarchus albifrons , a South American fresh water weak electric fish of the high 
’equency type (Figures 55, 5G). The electroreccptors of other species have to be 
.vestigated and the relationships of the various electroreceptors to the underwater 
ittern recognition system will' be established. 

3 mentioned the effect of moving objects past the electroreccptors in. one or another 
•ection. Some of them would respond in one way for the forward direct (head to tail) 
increasing the rale of the impulses and in another way for the backward direction 


Figure 55. Tonic, ampullary electroreceptor. 
Physical analog. 

current and voltage 



generator internal resistance 

external resistance 



autorhythmic generator 

external canal resistance 

r. • t. 
r il’ r i2 

internal resistance 

external canal capacitance 



internal receptor capacitance 

receptor inner face resistance 



skin resistance 



Figure 56 Phasic , tuberous electroreceptor. 

current and voltage 

Physical analog, 


generator internal resistance 

external resistance 



autorhythmic generator 

internal canal resistance 

r *l J r i2 

internal resistance 

internal canal capacitance 



internal receptor capacitance 

receptor cell capacitance 



skin resistance 

receptor inner face resistance C 



external skin capacitance 

(tall to head) by decreasing the rate of impulses. Other electroreccplors will react 
to higher or lower conductivity than the medium (aquarium water) in a similar way. 
Obviously, the response of the electrorcceptor.s in other electric fish species have to 
be assessed in order to get a better overall picture of the underwater pattern recogni- 
tion process. Figures 57 through 03 show two different elcctroreceptors of electric 
fish. The results obtained from the electric receptors study will lead to a 
plan of an underwater pattern recognition system. A passive hybrid underwater pattern 
recognition system block diagram is shown in Figure 64. In this block diagram beside 
the electric sensory receptors, the other types of lateralis line sensory receptors are 
combined to obtain a better cross -correlation of the different signals. Eventually 
passive optical sensor,- receptors will be added (for detecting, for example, M-py . 

in the bioluminescent organisms in the water when disturbed by a stimulus like heat, 
water turbulence, etc.). 

With regard to the other lateralis line organs, Sternarchus albifrons . like other teleosts, 
have canal organs as well as superficial organs, called free neuromasts. Some parti- 
cularities of these organs should be noted. Not all canal organs are in direct relation 
with the external environment. The sense organs do not always alternate with the pores 
cf the canal . In some cases, three sense organs are located between two pores. All 
three cupulae lie in a "watery" fluid filling the canal. The fluid seems to be a form of 
xjlymucosucharides. The canal is not free from one pore to another, but is obstructed 
)etween the front and second, and the second and third cupula-sense organ units by two 
jpithelial plugs. Thus, the middle unit is not in direct continuity with the external en- 
vironment (Ref. 23). It should be mentioned that Gymnarchus as well as Notopterus 
ave a completely closed canal system. The free neuromasts found in the head of 
te rnarchus are relatively large, are protected by paired prominent epidermal flaps 
nd have a histological structure similar to 'big pit organs". As in the pit organs of 
j gnna rebus (Ref. 25), these free organs of Sternarchus are innervated by a bundle 


.‘.:c57. Tonic Elcctrorcccptor South American Fresh Water Weak Electric Fish. 


FsFlATT£N'n> CELLS (kntt'SzdtoierlvJuMel) 

Figure 58. Phasic Electrorcceptor South American Fresh Water Weak Electric Fish. 



N „ A mill! 1 '! ltM-ww 

iTinmr. — 



AK>i-l »- 


— | 

iUUU-b* l 

1 iliil 



59. Ampullary tonic electroreceptor. 

Fig. 60 Stimulus and recording from 
an ampullary tonic electroreceptor. 

f,Ht i. 

61 Tuberous phasic clectrorcccplor. Fig. 62 Stimulus and recording from a 
(Schematic) , tuberous phasic electroreceptor. 


Out(!r ll/H 


ovtfr iL/«f 


Fig. 63. Electro receptors of Gvmnarchus niloticus . 

ampullary electroreceptor b: tuberous electroreceptor type b 

tuberous electroreceptor type c. (Schematic) 





o> —i 
> rt 

•rt D U 
B -H 8) 

3 % « 

ft O 




s § 

S w 
§ w 
W J 
co < 

o o 


S 3 

■*-» - W 

Hi ^ ft 

j w a 
w g Q 


ick and thin fibers. The specialized lateral line organs (electric sensory organs) 
be subdivided into two main groups: ampullary organs and tuberous organs. 

;e are many subdivisions of these organs and we have previously reported about 

different lateral line organ types, canal organs, free ncuromasts, ampullary 
ns and tuberous organs , have a characteristic distribution pattern being similar 
ymnotids, except for Electrophorus electricus . The density of each type of organ 
3 same species depends upon the size of the specimen, e.g. , canal organs in the 
region are separated by 1 mm in a Sternarchus albifrons 13 cm long, and by 2 mm 
e 22 cm long. 

ipproximate distribution of single organs was established by observing the "pore 
m" on the surface of the skin with a dissecting microscope. It follows from the 
ous descriptions of these organs that these "pores" should not be considered as 
1 hole in the integument in every case, but rather a local differentiation of the 
rmis overtying the sensory organs . 


ie was to develop the necessary instrumentation for microelectrode recordings 

iCtroreceptors of electric fish so as to be able to investigate their pattern 

ion ability. In the previous chapter we demonstrated that the system is work- 


and we succeeded in recording the autonomous autorhythmic electrical activity 
nic ampul lary electroreceptors of Stemarchus albifrons compared with the 
organ's normal activity (Figures 65 and 6G). 

arding was made during resting of the fish with no stimulus. The impulses 
■egular around a repetition rate of between 100 and 300. The amplitude was 
:. 5 mv per spike. The spike duration was very short — around 200 micro- 
(Figures 65, 66). 

r investigation, it can be concluded that electric fishes could use their elec- 
ms (transmitting and receiving) for navigation and communication — in other 
pattern recognition. 

mdings and histological evidence show that Sternarchus albifrons has three 
electroreceptors : ampullary tonic nonsynchronous units , ampullary tonic 
ious units, and tuberous phasic nonsynchronous units. The physical analogs 
md phasic electroreceptors are shown in Figures 57 and 58. Both are repre- 
7 a generator connected to resistances and capacitances in series and in 
The difference between tonic and phasic electrorcceptors is that the first 
e one resistance in scries with the generator whereas the phasic electro- 
s have a capacitance. The tonic clectroreceptors seem to be predominant, 
ke five-to-one, compared to the phasic electroreceptors. The elcctrore- 
seem to act, to a certain extent, independently of the main electric transmitting 
least two out of three different types of electro receptors are asynchronous 

Figure 65. Recording from an 
anaesthetized, curarizcd Ste marches 
albifrons specimen. Horizontal: 

1 graduation = 1 msec. Vertical: 

1 graduation = 10 mV. 

igure 66. Microelcctrodc recording of 
le autorhythmic electrical activity of 
ic ampulla ry, tonic electroreceptors of 
ternarchus albifrons. The spikes seen 
n the top of the rhythmic almost 
inusoidal waveform are the electric 
ignals from the electrorcceptors. 
orizontal: 1 graduation = 2 msec, 
'ertical: 1 graduations 500 mV. 
mplification xlOO, effective 1 gradua- 
ion = 5 mV. Spike app. 2 to 2.5 mV. 


ne type of electro receptor will synchronize with jhe main electric organ, 
ound that the complete denervation of the transmitting electric organ does 
e activity of the asynchronous electroreceptors (both pluisic and tonic). The 
i capable of responding to conductive and nonconductive objects placed near 
»dy. It may affect the total capability in determining certain movements or 
a certain extent, its sensitivity in pattern recognition. Some of the syn- 
mic units are connected to one and the same nerve trunk part of the acoustico 
/stem but connected to specialized big nuclei in the brain. 

triking fact about fresh water weak electric fish, besides their spontaneous 
jan, is that all of them are provided with a highly developed lateralis line 
elated to tins acoustico-lateralis system is an enlargement of the cere- 
leciaHy in Gymnarchus niloticus_ and in mormyrids. The unusual importance 
ilis system in these fish, compared with other teleosts, is not due to an in- 
nber of "ordinary" lateral line sensory organs, but rather to the existence 
umber of specialized sensory organs within this same system. 

>orting our hypothesis about a hybrid complex underwater pattern recognition 
1 by electric fishes in recognition of prey, predators, and navigation in 
is recommended that the other lateralis line systems from different fresh 
electric fishes should be studied with the aim to find out the role of the 
isory organs in pattern recognition. 

t of the electric fish pattern recognition system would make it possible to 
models of the physical analogs of the sensors could be integrated in object 
location, detection and identification. The range and sensitivity of the 
i be assessed and improvements could be made. 

loly used anaesthetic,- "MS 222" or "Finqucl" (tricaiue mcthancsulfonatc) 
•cis the repetition rale of the electric impulses of the electric' organ. It 
10 do a series of experiments on different anaesthetics to establish whether 

:e is one which would not affect the frequency of the impulses. It has been found 
; thiopental sodium (sodium penthotal) will not affect the frequency of the impulses 
is a safe anaesthetic for fish, acting fast and without any ill effects. 

er subdivisions exist between the one and the same type of electroreceptor, but this 
not been as yet investigated in a detailed way. The connections between the electro- 
eptors, the different nerves, nerve -trunks and the brain have to be investigated. In 
; way, their interrelationship could be established. Microelectrode recordings from 
electroreceivers proper and from their nerve fibers have been planned. Electro- 
section and clearing of the lateral line near the electroreceptors to be investigated 
. enable us to record from the efferent nerve fibers. 

m the experiments with Gymnarchus niloticus, we concluded -that one fish would 
agnize and communicate with another one of the same species. Behavioral experi- 
lts in this direction will be continued. It is recommended that further micro- 
rtrode recordings be made from the electroreceptors and from the nerve fibers of 
marchus albifrons and of the newly received Gymnarchus niloticus . 

3 possible to simulate an equivalent sensory system responding to different stimuli 
erwater. A system with a double feedback mechanism can be envisaged: (1) one 
resented by a constant frequency electric field transmitting system operating on 
phase -synchronous electroreceptors responding to discontinuities in the electric 
d or to changes in the phase relationship transmitter -receptor; and (2) another 
represented by a variable frequency transmitting system responding to disturbances 
he field between transmitter and receptor with a change of the frequency of the 
ismitting electric organ. To this we could add an independent dual autorhythmic 
eptor system: (a) responding with the increase or decrease of the autorhythmic 
luency depending on movement direction of the disturbance in the electric field; 

(b) responding with a change in the latency depending on the magnitude of the dis- 
tance, also distinguishing between conductive and nonconduetive objects. 

rrmarchus niloticus , one of the most sensitive electric fishes, served as a model for 
simulation of a simple pattern recognition system (Figures 67 and 68). 

«• •• 


Fig. 69 Laboratory for investigation of electric fishes A. 


Watanabe, A., andK. Takeda. J. Exn. Biol. 40:57 (1963). 

Bennett, M. V. , Modes of Operation of Electric Organs. Annals of the New 
York Academy of Science 94:2:458 (1961). 

Eccles, J. C., P. Fatt, andK. Koketsu, J. Physiol. 126:524 (1954). 

Rose, J. E., and V. B. Mountcastle, Bull. Join Hopkins Hosp. 94:23S (1954). 

Grundfest, H. , Progress in Biophysics 7:1-85. Pergamon Press, London, 
G.B. (1957). 

Furukawa, J. , and Furshpan, E. J. , Two inhibitory mechanisms in the 
Mauthner neurons of goldfish, J. Neurophysiol . 26:140-176 (1963). 

Diamond, J. , and Yasargil, G. M. , Synaptic function in the fish spinal cord, 
Dendritic integration. Prog. Brain Res. 31:201-209 (-1969). 

Auerbach, A. A. and Bennett, M.F.L. , Chemically mediated transmission 
at a giant fiber synapse in the central nervous system of a vertebrate, J. Gen . 
Physiol. 53:183-210 (1969). 

Auerbach, A. A. and Bennett, M.V.L. , A rectifying synapse in the central 
nervous system of a vertebrate. J. Gen. Physiol. 53:211-237 (1969). 

Agalidcs, E. and Sparks, S. , The compound eye of arthropods in "Research 
and Development" GDPD General Dynamics, pp. 317-322 (1968). 

Hartlinc, H. K. , Wagner, II. G. , and Ratcliff,. F. , Inhibition in the eye of 
Limulus . J. Gen. Phvsiol. 39:651-673 (1956). 

Lissman, II. W., and Mullinger, A. M. , Organization of ampullary electric 
receptors in Gymnotidac (Pisces), Proc. Roy. Soc. 31G9:315-37S (19GS). 

Szabo, T. , Sense organs of the lateral line system in some electric if the 
Gymnotidac , lUormvridac , and Gymnarchidac. J. Morphol. 117:220-250 (1965). 

6. Szamier, R. B. and Wachtel, A. W. , Special cutaneous receptor organs of 

fish. VI. The tuberous- and ampullary organs of Hypopomus. J. Ultras! met. 
Res . 30:450-471 (1970). 

.7. De Olive ria Castro, G. , Morphological data on the brain of Elcctropliorus 

electricus (L) in Bioelectrogenesis (C. Chagas and A. P. de Carvalho, eds.), 
pp. 171-182, Elsevier, Amsterdam (1961). 

18. Bennett, M. V. L., Mechanism of electi*o reception. In "Lateral Line De- 
tectors” (P. Cahn, ed.), pp. 313-393, Indiana Uni v. Press, Bloomington, 
Indiana (196 7) * 

19. Suga, N.', Electrosensitivity of canal and free neuromast organs in a gymnotid 
electric fish. J. Comp. Neurol. 131:453-458 (1967). 

20. Bennett, M.V.L. , Similarities between chemically and electrically mediated 

transmission. In "Physiological and Biophysical Aspects of Nervous Inte- 
gration." (F.D. Carlson, ed.) pp. 78-128, Prentice Hall, Englewood Cliffs, 
New Jersey (1968). • 

21. Mullinger, A. M. , The organization of ampullary sense organs in the electric 
fish Gymnarchus niloticus , Tissue Cell 1:31-52 (1969). 

22. Lissman, H. W., andK. E. Machin, J. Exp. Biol. 35:451-486 (1958). 

23. Bullock, T. H. Personal communication (1969). 

24. Szabo, T. and Fessard, A. E., J. Physiol. , Paris. 57:343-360(1965). 

25. Bullock, T. H. and Chichibu, S. , Proc. National Acad. Sci. USA. 54:422- 
429 (1965). 

26. Agalides, E. Evolution of electric organs in fish, information processing by 
electric fish and communication between electric fish. Lecture at the Arthur 
D. Little Co. , Cambridge, Mass. (21 Feb. 1961). 

27. Agalides, E., D. HeppncrandC. Claysmith. Communication techniques to 
resist jamming. GDCA-ERR-1717, Convair Aerospace Div. (October 1972). 

28. Hubei, D. H., Sciences, Vol. 125, pp. 549-550 (1957). 


29. Wolhbarsht, M. L. , MacNichol, E. F. , Jr., and Wagner, II. G., Science , 
Vol. 132, pp. 1309-1310 (1960). 

3 rundfest, II. , Scngstakcn, R. W. f Oettinger, W. H 
?ov. of SrT- Inslr. . Vol. 21, pp. 360-361 (1950). 

and Gurry, R. W. , 

Donaldson, P.E.K., Electronic Apparatus for Biological Research, Butter 
vorthsSci. Publ. , London, pp. 568-581 (1958). 

Vood, E. M. , Urethane as a carcinogen, 
lo. 3 (1956). 

Progressive Fish-Culturist, Vol. 18, 

3all, J. N. , and Cowen, P. N. , Urethane as a carcinogen and as an anaes- 
hetic for fishes. Nature, Vol. 184, p. 370 (1959). 

love, F. J. MS-222 Sandoz, the anaesthetic of choice for fish and other cold 
dooded organisms. Sandoz News No . 3 (1965). 

Marking, L. L. , Investigations in Fish Control: 12. 
selected fishes . U.S. Bureau of Sport Fisheries and 
ication No. 18 (1966). 

ToxL ity of MS -222 to 
Wildlife Resource Pub' 

Valker, C. R. , mi Sohoettger, R. A. , Investigations in Hsh Control: Resi- 
iues of MS-222 in four species of salmonids follo'ving anesthesia. JJ, f S. Bur g, 
,f Sport Fisheries and Wildlife Resource Publication.No. 21 (1966). 

'choett-er R. A. , and Julin, A. M. , 13. Efficacy of MS-222 as an Anesthetic 

Fo« Ssimonids . UJ, B ureau of -port Fineries and Wildlife ResQurss. 
publication No . 19 (1967). 

Gontz G. W. , "Anesthesia of Fishes”, in Experimental 
iy D. C. Sawyer, AD 648 329, U.S. Dept, of Commerce 
Dec. 1964 at the Brooks AFB, Texas. 

Animal Anesthesiology 
1967 Symposium held 


zabo. Th., in Tnteral Line Detectors (P. Cahn, ed.>, pp. 295-311. Indiana 
Jniversity Press (1971). 

laghvara. S. . Szaho, Th. , and Engor, P.S..J.. NcurophysioT 28:775-733 (1905) 
’.nger,' P.S. , and Szabo. Th. , .1. of Neurophysiol. 28:775-783 (1905) 

Tnbna Ruga.. J. of Comp. Neurol. 131:4:437-451 (1967). 

>ullock, T. II., Brain Behav. Evol. 2:85-118 (1969) 

Iartop: U. S. Patent 3109001 to Abbot Labs (1965). 

Vgalides , E. , Sensitivity and Behavioral Reaction of Sharks to Electric 
Stimuli, Final Report. ONR Contract Nonr 4773(00) No. 104-8G3 (1967). 

lagiwara, S. , Szabo, T., and Engcr, P.S., Electroreceptor mechanism in 
i high frequency wealdy electric fish, Stemarchus albifrons. J. Neurophysiol. 
28:5:784-799 (1965). 

Szabo, T. , Nature, 194:4843:600-601 (1962).